Background Not all breast cancer (BC) patients who receive neoadjuvant chemotherapy achieve a pathologic complete response (pCR), but the reasons for this are unknown. Previous studies have shown that exosomes produced in the tumor microenvironment in response to chemotherapy promote a chemotherapy-resistant phenotype in tumors. However, the role of BC chemotherapy-elicited exosomes in regulating chemoresistance is poorly understood. Methods Using commercial kits, serum exosomes were extracted from patients before neoadjuvant chemotherapy, after one cycle of chemotherapy and after four cycles of chemotherapy consisting of doxorubicin (DOX) and paclitaxel (PTX). Their miRNAs were sequenced, and the correlation between the sequencing results and chemotherapy effects was further verified by RT-qPCR using patient serum exosomes. Cell Counting Kit-8 (CCK-8) was used to detect chemosensitivity. Stemness was assessed by CD44+/CD24- population analysis and mammosphere formation assays. Chromatin immunoprecipitation (ChIP) experiments were performed to verify the binding of signal transducer and activator of transcription 3 (STAT3) to the promoter of miRNAs. Results Here, we provide clinical evidence that chemotherapy-elicited exosomal miR-378a-3p and miR-378d are closely related to the chemotherapy response and that exosomes produced by BC cells after stimulation with DOX or PTX deliver miR-378a-3p and miR-378d to neighboring cells to activate WNT and NOTCH stemness pathways and induce drug resistance by targeting Dickkopf 3 (DKK3) and NUMB. In addition, STAT3, which is enhanced by zeste homolog 2 (EZH2), bound to the promoter regions of miR-378a-3p and miR-378d, thereby increasing their expression in exosomes. More importantly, chemotherapeutic agents combined with the EZH2 inhibitor tazemetostat reversed chemotherapy-elicited exosome-induced drug resistance in a nude mouse tumor xenograft model. Conclusion This study revealed a novel mechanism of acquired chemoresistance whereby chemotherapy activates the EZH2/STAT3 axis in BC cells, which then secrete chemotherapy-elicited exosomes enriched in miR-378a-3p and miR-378d. These exosomes are absorbed by chemotherapy-surviving BC cells, leading to activation of the WNT and NOTCH stem cell pathways via the targeting of DKK3 and NUMB and subsequently resulting in drug resistance. Therefore, blocking this adaptive mechanism during chemotherapy may reduce the development of chemotherapy resistance and maximize the therapeutic effect.

Misregulation of BCL-2 family of proteins renders a survival signal to withstand cytotoxic anticancer drugs and is often found in drug resistant cells. The drug resistance phenotype is also associated with an enhancement of cancer stem cell-like (CSC) characteristics. Thus, inhibition of anti-apoptotic BCL-2 family proteins has been proposed as a possible antineoplastic strategy, and BCL-2 inhibitors are currently being clinically trailed in patients with leukemia, lymphoma or non-small cell lung cancer. However, the effects of BCL-2 inhibitors on drug resistant breast cancer have not yet been elucidated. In the present study, the effect of sabutoclax, a pan-active BCL-2 protein family antagonist, on two chemoresistant breast cancer cell lines was assessed. We found that sabutoclax showed a significant cytotoxic activity on chemoresistant breast cancer cells both in vitro and in vivo. When chemotherapeutic agents were combined with sabutoclax, strong synergistic antiproliferative effects were observed. Sabutoclax induced the blockage of BCL-2, MCL-1, BCL-xL and BFL-1, which in turn led to caspase-3/7 and caspase-9 activation and modulation of Bax, Bim, PUMA and survivin expression. Furthermore, sabutoclax effectively eliminated the CSC subpopulation and reduced sphere formation of drug-resistant cells through down-regulation of the IL-6/STAT3 signaling pathway. A similar effect was observed in a small panel of nine breast tumors ex vivo. Our findings indicate that sabutoclax partially overcomes the drug resistance phenotype of breast cancer cells by reactivation of apoptosis, mediated by the inhibition of several anti-apoptotic BCL-2 family proteins, and eliminates CSCs by abolition of the IL-6/STAT3 pathway. This offers a strong rationale to explore the therapeutic strategy of using sabutoclax alone or in combination for chemotherapy-nonresponsive breast cancer patients.

Triple-negative breast cancer (TNBC) refers to a heterogeneous group of tumors, for which there is currently a lack of targeted therapies. Poly(ADP-ribose) polymerase (PARP) inhibitors, phosphatidylinositol 3-kinase (PI3K) inhibitors and carboplatin (CBP) have demonstrated sufficient efficacy and safety for their use as individual drugs for the treatment of TNBC; however, their effects on TNBC when used as a combination have not been investigated. The primary objectives of the present study were to determine the effects of a combination of CBP, olaparib and NVP-BKM120 (BKM120), and to investigate the mechanism underlying their effects on TNBC cells. The drug combination was cytotoxic to TNBC cells, both with regards to short-term and long-term sensitivity, as determined using colony forming assays, and they exerted strong synergistic effects on MDA-MB-231 and CAL51 cell lines. All drugs affected cell cycle progression, and western blotting and immunofluorescence indicated that the the drug combination exerted its cytotoxicity via DNA damage, enhancing non-homologous end joining repair and inhibiting homologous recombination repair. These data provide a strong rationale to explore the therapeutic use of olaparib in combination with CBP and BKM120 in animal models, and later in clinical trials on patients with TNBC.

No abstract